Techniques for the Biomedical Sciences - Essay Example

Laboratory Techniques for the Biomedical Sciences Introduction ELISA tests have been widely used in separation of antibodies especially by the use of blood serum. They are preferable since they can handle a rapid large number of samples in parallel. ELISA was developed in 1970 as a replacement for radioimmunoassay. ELISA is a biochemistry assay that is wet in nature used in detection of substance usually an antigen. Results and Discussion 1. The plates are washed in the first place to sterilize any microorganism that may be present that may alter the results of the experiment. 200 ul of 3% Marvel in PBS-Tween is left for 45 minutes at room temperature to allow the serum to settle well in the plates. This is the blocking process which contains either the antigen or the antibody. The liquid is attached to the solid creating a thin film layer. The wells are washed to remove any serum that does not bind with plate. Excess liquid is removed during the process using a blotting paper to avoid dilution of solutions used in the next stage. Wells 1A, 3A, and 4A are added with different concentration of 100ul of hCG serum (100 ng ml-1) in order to come up with varied results which are not uniform. Extracts from well 1A is thoroughly mixed with 1B to 1F. This is done to attach the serum with specific antigen or antibody. The resulting solution is incubated at room temperature for 45 minutes to allow the substrates and serum to mix thoroughly. Washing is done to remove any protein that did not attach itself with the substrate. 150ul p-nitrophenyl phosphate substrate is added in each well in order for the substrate to attach to the serum. The substrate contains specific enzyme that is used for identification. The yellow colors indicate that the results are positive [Jan01]. While conducting ELISA tests mild detergents are normally used in washing out the proteins that are not bound to any particular substrate. ELISA has been widely used in pregnancy tests. Chorionicgonadotrophin (hCG) is a hormone largely produced by developing placenta during pregnancy [Roi01]. Peptide hormone detection is used in pregnancy tests and many other tests such as abnormally growing cell tumors most importantly the germ cell tumors. Chorionicgonadotrophin (hCG) hormone is also useful in man in determination of cancer of the testicles. 2. ELISA Practical results Row 1 2 3 4 A 0.06 0.048 0.009 0.748 B 0.048 0.055 0.01 0.756 C 0.06 0.042 0.006 0.578 D 0.031 0.063 0.008 0.214 E 0.047 0.054 0.006 0.143 F 0.055 0.048 0.007 0.097 All absorbances at 410nm. Row 1 2 3 4 A 10 ng 0 10 ng 10 ng B 5 ng 0 5 ng 5 ng C 2.5 ng 0 2.5 ng 2.5 ng D 1.25 ng 0 1.25 ng 1.25ng E 0.63 ng 0 0.63 ng 0.63 ng F 0.31 ng 0 0.31 ng 0.31 ng 3. A typical graph plotted will assume a sigmoid shape. This is mainly attributed to the wells from column 4 which indicate a yellow colour showing positive results. However, there is increased absorption as the concentration increases. Wells that did not contain the serum showed little or no absorbance. For the wells which did no have the substrate (3A-3F) indicated the least absorbance. 4. Sandwich ELISA This type of ELISA has been known to require the use antibody pairs which are matching. In this case, the antibody is first coated in the pate well by an antigen molecule. The next solution known as analyte is then applied to the well. This is then followed by addition of another antibody layer, which helps in measuring the concentration of the analyte. Capturing and detection of analyte is possible through use of polyclonals in sandwich ELISA is variability is allowed. In cases where the enzyme is conjugated with the detecting antibody, the process is known as direct sandwich ELISA [Rud09]. However, if the antibody in reference is unknown another antibody is usually required hence, the process is referred to as indirect ELISA. The false positive and negative results can be avoided by ensuring that the window period is well observed to reduce the errors. 5. Western blot It is similar to ELISA test but the difference is the immobilization and separation of viral proteins. From this step, binding occurs where the specific serum is attached to specific HIV protein. Most of the western bolt is sold commercially with HIV proteins already applied. Onc the proteins are well laid on the wells an electrical current is allowed to pass through them. This enables them to flow at different velocities leading to separation [JMA06]. From this stage, the procedure follows that of an ELISA. This process involves application of human antibody through the blood. The serum that does not attach is washed out. For the results to be positive one of this viral bands must be present. GAG, POL, and ENV. If they are missing, the result is negative. 6. = log [-7] M = 10-7 x 1000 mM = 10-4 mM Log -5= 0.6989 = log [-8.80] M = 10-8.8 x 1000 mM = 10-5.8 mM Antibody reactions mostly use avidity where the multiple antigen-binding sites are interacting with the specific substrate. This interaction may also lead to individual binding being broken down [Cho05]. The process of unbinding does no lead to the molecule diffusing away. Affinity involves measuring the strength of binding. High interaction affinities regenerated by hydrogen bonds, electrostatic bonds, hydrophobic bonds and many others. Igm is normally assumed to be having low affinity but usually having a high avidity since it possess 10 weak binding sites. Bibliography Jan01: , (Janeway CA Jr, 2001), Roi01: , (Roitt, 2001), Rud09: , (Rudnick SI, 2009), JMA06: , (Appel, 2006), Cho05: , (Chou R, 2005), Read More